A dual-acting DNASE1/DNASE1L3 biologic prevents autoimmunity and death in genetic and induced lupus models.

A defining feature of systemic lupus erythematosus (SLE) is loss of tolerance to self-DNA, and deficiency of DNASE1L3, the main enzyme responsible for chromatin degradation in blood, is also associated with SLE. This association can be found in an ultrarare population of pediatric patients with DNASE1L3 deficiency who develop SLE, adult patients with loss-of-function variants of DNASE1L3 who are at a higher risk for SLE, and patients with sporadic SLE who have neutralizing autoantibodies against DNASE1L3. To mitigate the pathogenic effects of inherited and acquired DNASE1L3 deficiencies, we engineered a long-acting enzyme biologic with dual DNASE1/DNASE1L3 activity that is resistant to DNASE1 and DNASE1L3 inhibitors.

Quantitative correlation of ENPP1 pathogenic variants with disease phenotype.

Ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) codes for a type 2 transmembrane glycoprotein which hydrolyzes extracellular phosphoanhydrides into bio-active molecules that regulate, inter alia, ectopic mineralization, bone formation, vascular endothelial proliferation, and the innate immune response. The clinical phenotypes produced by ENPP1 deficiency are disparate, ranging from life-threatening arterial calcifications to cutaneous hypopigmentation. To investigate associations between disease phenotype and enzyme activity we quantified the enzyme velocities of 29 unique ENPP1 pathogenic variants in 41 patients enrolled in an NIH study along with 33 other variants reported in literature.

Outer hair cell function is normal in βV spectrin knockout mice.

Reports have proposed a putative role for βV spectrin in outer hair cells (OHCs) of the cochlea. In an ongoing investigation of the role of the cytoskeleton in electromotility, we tested mice with a targeted exon deletion of βV spectrin (Spnb5), and unexpectedly find that Spnb5 animals' auditory thresholds are unaffected. Similarly, these mice have normal OHC electromechanical activity (otoacoustic emissions) and non-linear capacitance.

Strategies for Glycoengineering Therapeutic Proteins.

Almost all therapeutic proteins are glycosylated, with the carbohydrate component playing a long-established, substantial role in the safety and pharmacokinetic properties of this dominant category of drugs. In the past few years and moving forward, glycosylation is increasingly being implicated in the pharmacodynamics and therapeutic efficacy of therapeutic proteins. This article provides illustrative examples of drugs that have already been improved through glycoengineering including cytokines exemplified by erythropoietin (EPO), enzymes (ectonucleotide pyrophosphatase 1, ENPP1), and IgG antibodies (e.

Musculoskeletal Comorbidities and Quality of Life in ENPP1-Deficient Adults and the Response of Enthesopathy to Enzyme Replacement Therapy in Murine Models.

Ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) deficiency leads to cardiovascular calcification in infancy, fibroblast growth factor 23 (FGF23)-mediated hypophosphatemic rickets in childhood, and osteomalacia in adulthood. Excessive enthesis mineralization and cervical spine fusion have been previously reported in patients with biallelic ENPP1 deficiency, but their effect on quality of life is unknown. We describe additional musculoskeletal complications in patients with ENPP1 deficiency, namely osteoarthritis and interosseous membrane ossification, and for the first time evaluate health-related quality of life (HRQoL) in patients with this disease, both subjectively via narrative report, and objectively via the Brief Pain Inventory-Short Form, and a Patient Reported Outcome Measurement Information System Physical Function (PROMIS PF) short form.

Improving the Pharmacodynamics and In Vivo Activity of ENPP1-Fc Through Protein and Glycosylation Engineering.

Enzyme replacement with ectonucleotide pyrophosphatase phospodiesterase-1 (ENPP1) eliminates mortality in a murine model of the lethal calcification disorder generalized arterial calcification of infancy. We used protein engineering, glycan optimization, and a novel biomanufacturing platform to enhance potency by using a three-prong strategy. First, we added new N-glycans to ENPP1; second, we optimized pH-dependent cellular recycling by protein engineering of the Fc neonatal receptor; finally, we used a two-step process to improve sialylation by first producing ENPP1-Fc in cells stably transfected with human α-2,6-sialyltransferase (ST6) and further enhanced terminal sialylation by supplementing production with 1,3,4-O-Bu ManNAc.

Clinical and Biochemical Phenotypes in a Family With ENPP1 Mutations.

Inactivating mutations of the ENPP1 gene are associated with generalized arterial calcification of infancy (GACI) and less often autosomal-recessive hypophosphatemic rickets type 2 (ARHR2). We aimed to investigate the spectrum of phenotypes in a family with monoallelic and biallelic mutations of ENPP1 after identification through whole exome sequencing of a 54-year-old female with biallelic mutation of ENPP1, c.323G > T; p.

Human Heterozygous ENPP1 Deficiency Is Associated With Early Onset Osteoporosis, a Phenotype Recapitulated in a Mouse Model of Enpp1 Deficiency.

Biallelic ENPP1 deficiency in humans induces generalized arterial calcification of infancy (GACI) and/or autosomal recessive hypophosphatemic rickets type 2 (ARHR2). The latter is characterized by markedly increased circulating FGF23 levels and renal phosphate wasting, but aberrant skeletal manifestations associated with heterozygous ENPP1 deficiency are unknown. Here, we report three adult men with early onset osteoporosis who presented with fractures in the thoracic spine and/or left radius, mildly elevated circulating FGF23, and hypophosphatemia.

Cell organization, growth, and neural and cardiac development require αII-spectrin.

Spectrin α2 (αII-spectrin) is a scaffolding protein encoded by the Spna2 gene and constitutively expressed in most tissues. Exon trapping of Spna2 in C57BL/6 mice allowed targeted disruption of αII-spectrin. Heterozygous animals displayed no phenotype by 2 years of age.

Ankyrin recognizes both surface character and shape of the 14-15 di-repeat of beta-spectrin.

The spectrin-based cytoskeleton is critical for cell stability, membrane organization and membrane protein trafficking. At its core is the high-affinity complex between beta-spectrin and ankyrin. Defects in either of these proteins may cause hemolytic disease, developmental disorders, neurologic disease, and cancer.

The structure of the ankyrin-binding site of beta-spectrin reveals how tandem spectrin-repeats generate unique ligand-binding properties.

Spectrin and ankyrin participate in membrane organization, stability, signal transduction, and protein targeting; their interaction is critical for erythrocyte stability. Repeats 14 and 15 of betaI-spectrin are crucial for ankyrin recognition, yet the way spectrin binds ankyrin while preserving its repeat structure is unknown. We have solved the crystal structure of the betaI-spectrin 14,15 di-repeat unit to 2.

Ankyrin facilitates intracellular trafficking of alpha1-Na+-K+-ATPase in polarized cells.

Defects in ankyrin underlie many hereditary disorders involving the mislocalization of membrane proteins. Such phenotypes are usually attributed to ankyrin's role in stabilizing a plasma membrane scaffold, but this assumption may not be accurate. We found in Madin-Darby canine kidney cells and in other cultured cells that the 25-residue ankyrin-binding sequence of alpha(1)-Na(+)-K(+)-ATPase facilitates the entry of alpha(1),beta(1)-Na(+)-K(+)-ATPase into the secretory pathway and that replacement of the cytoplasmic domain of vesicular stomatitis virus G protein (VSV-G) with this ankyrin-binding sequence bestows ankyrin dependency on the endoplasmic reticulum (ER) to Golgi trafficking of VSV-G.

Stimulation of Galphaq-coupled M1 muscarinic receptor causes reversible spectrin redistribution mediated by PLC, PKC and ROCK.

Spectrin is a cytoskeletal protein that plays a role in formation of the specialized plasma membrane domains. However, little is known of the molecular mechanism that regulates responses of spectrin to extracellular stimuli, such as activation of G-protein-coupled receptor (GPCR). We have found that alphaII spectrin is a component of the Galpha(q/11)-associated protein complex in CHO cells stably expressing the M1 muscarinic receptor, and investigated the effect of activation of GPCR on the cellular localization of yellow-fluorescent-protein-tagged alphaII spectrin.

Human Sec31B: a family of new mammalian orthologues of yeast Sec31p that associate with the COPII coat.

We have cloned human brain and testis Sec31B protein (also known as secretory pathway component Sec31B-1 or SEC31-like 2; GenBank accession number AF274863). Sec31B is an orthologue of Saccharomyces cerevisiae Sec31p, a component of the COPII vesicle coat that mediates vesicular traffic from the endoplasmic reticulum. Sec31B is widely expressed and enriched in cerebellum and testis.

Expression of ZER6 in ERalpha-positive breast cancer.

Background: ZER6 is a C2H2 zinc finger transcription factor with two isoforms (p52-ZER6 and p71-ZER6), which are differentially repressed by a ligand-dependent interaction with estrogen receptor-alpha (ERalpha). We sought to determine if ZER6 proteins are expressed in ERalpha-positive breast cancer cells and if ZER6 is expressed in association with ERalpha in breast cancers.

Methods: The expression of ZER6 protein was examined by Western blot and the pattern of ZER6 expression was examined in a panel of ERalpha-positive and ERalpha-negative breast cancers using RT-PCR.